S J WakilG M Mabrouk, I M Helmy, K G Thampy and  and epinephrine.carboxylase. The roles of insulin, glucagon, Acute hormonal control of acetyl-CoA:

1990, 265:6330-6338.J. Biol. Chem. 

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THE JOURNAL OF BIOLWXCAL CHEMISTRY Vol. 265, No. 11, Issue of April 15, pp. 633%6338,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in Il. S. A.

Acute Hormonal Control of Acetyl-CoA Carboxylase THE ROLES OF INSULIN, GLUCAGON, AND EPINEPHRINE*

(Received for publication, October 13,1989)

Gamal M. MabroukS, Ihab M. Helmy$, K. George Thampyg, and Salih J. Wakiln From the Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given O-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. The carboxylase from untreated rats has rel- atively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/m01 of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/m01 of subunit). Addition of citrate activates both preparations with half-maximal activation (&.s) at 1.0 and 0.6 mM citrate, respec- tively. The enzyme from rats that did not receive in- sulin is mainly in the octameric state (M= - 2 x 106), while that from rats that received insulin is mainly in the polymeric state (iUr - 10 x 10’). Thus, short-term administration of insulin results in activation of acetyl- CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the pro- tein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphory- lated by the Mn2+-dependent [acetyl-CoA carboxylase] -phosphatase 2.

The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the ab- sence of citrate), lower phosphate content (4.9 mol of Pi/m01 of subunit), and is present mainly in the poly- meric state (M= - 10 x 106). Addition of citrate acti- vates the enzyme with &.s = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/m01 of subunit, respectively), and mainly in the octameric state (Mr - 2 x 106). Treatment of these preparations with [acetyl-CoA carboxylasel-phosphatase 2 reacti-

* This work was supported in part by Grant DK-35419 from the National Institutes of Health and by a grant from The Clayton Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Biochemistry, Ain Shams University, Cairo, Egypt.

§ Present address: Dept. of Biochemistry, Indiana University School of Medicine, Fort Wayne Center for Medical Education, 2101 Coliseum Blvd., East, Fort Wayne, IN 46805.

11 To whom correspondence should be addressed: Verna and Marrs McLean Dept. of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-4528.

vated the enzyme (specific activity -8 unitslmg protein in the absence of citrate) and polymerized the protein (A!& - 10 x 106).

These observations indicate that insulin and gluca- gon, by altering the phosphorylation state of the acetyl- CoA carboxylase, play antagonistic roles in the acute control of its activity and therefore in the regulation of fatty acid synthesis.

Acetyl-CoA carboxylase plays an important role in the metabolism of fatty acids. It catalyzes the committed step in fatty acid synthesis, producing malonyl-CoA, the donor of the CZ units for the synthesis of long-chain fatty acids (l-3). Malonyl-CoA is also the allosteric regulator of the carnitine palmitoyltransferase shuttle system responsible for trans- porting fatty acyl groups through the mitochondrial mem- brane and making them available for oxidation by the /3- oxidation enzymes (4). Hence, investigations of the structure- function of the carboxylase and its regulation become essen- tial to our understanding of the process of fatty acid metab- olism, especially since the latter plays a pivotal role in the energy metabolism of vertebrates. The carboxylase is under both long-term control, involving changes in its mRNA levels and in the rate of protein synthesis and degradation (5-7), and short-term control, involving allosteric modification by citrate and palmitoyl-CoA and covalent modification by phos- phorylation. The physiological significance of these modifi- cations in regulating the activity of acetyl-CoA carboxylase has been unclear and controversial (8-12). Citrate is a re- quired activator of animal acetyl-CoA carboxylase with half- maximal activation (I&,) observed at concentrations of 2-10 mM (13-17), which are significantly higher than intracellular concentrations of 0.17-0.45 mM (18) and may not be an indicator of the rate of fatty acid synthesis (19).

Phosphorylation/dephosphorylation of the carboxylase has presented another problem. The isolated enzyme is a phos- phoprotein containing 6-15 mol of Pi/m01 of subunit (20,21); little is known about the kinase(s) and phosphatase(s) that are specific for acetyl-CoA carboxylase. The role of the car- boxylase-specific kinase(s) and phosphatase(s) in regulating enzyme activity remains ambiguous, with several reports in- dicating a direct relationship between phosphorylation and catalytic activity (8, 9, 22-30), while others could not dem- onstrate such an effect (10-12).

Diet, especially a fat-free diet, induces the synthesis of acetyl-CoA carboxylase and increases its activity. Starvation or diabetes represses the expression of the carboxylase gene and decreases the activity of the enzyme. Although not much is known about the regulation of the carboxylase gene, our recent studies involving dietary manipulations in rats have suggested an interrelationship between catalytic activity, cit- rate requirement, phosphorylation state, and polymerization

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Hormonal Control of Acetyl-CoA Carboxylase 6331

of acetyl-CoA carboxylase (31). Many studies aimed at un- derstanding the effect of hormones (insulin, glucagon, epi- nephrine, and the like) on acetyl-CoA carboxylase have yielded confusing and contradicting results. Although insulin is known to stimulate fatty acid synthesis while glucagon antagonizes its effects, both hormones have been reported to induce phosphorylation of acetyl-CoA carboxylase in dis- rupted tissues or cells (32-37). While there is a general con- sensus about glucagon-induced phosphorylation and inacti- vation of carboxylase (35,37,38), there is much less agreement on the effects of insulin, even among reports from the same laboratory. For instance, Witters et al. (39) have suggested that insulin induces dephosphorylation of acetyl-CoA carbox- ylase in hepatocytes. However, later studies showed that insulin induces phosphorylation by increasing 32P incorpora- tion into carboxylase of rat epididymal fat pads (32,34); and, of late, dephosphorylation of the enzyme by decreasing 32P labeling after exposure of Fao-Reuber hepatoma cells to in- sulin (40). Also, several investigators have reported insulin- induced phosphorylation of carboxylase with either no accom- panying activation (32,41), with activation (33,34,42-44), or with activation and polymerization (45). In some of these studies the insulin-induced phosphorylation survived purifi- cation of the carboxylase; the activation did not (34, 43). These studies led Ha&e’s group (43) to suggest that although insulin stimulates phosphorylation, the accompanying acti- vation of carboxylase is unrelated and may be due to the presence of an insulin-induced, and as yet unidentified, allo- steric activator.

Dephosphorylation of acetyl-CoA carboxylase by an [ace- tyl-CoA carboxylasel-phosphatase isolated from rat epididy- ma1 fat pads resulted in its activation (8). The phosphatase has broad substrate specificity and does not require a metal ion. More recently, however, a Mn*+-dependent phosphatase ([acetyl-CoA carboxylase] -phosphatase 2) has been purified from rat liver and shown to activate the carboxylase about lo-fold (13). This phosphatase has high affinity for the car- boxylase and renders it citrate-independent, when compared to the phosphorylated form of the carboxylase that is citrate- dependent. Moreover, dephosphorylation of the carboxylase by this phosphatase results in its polymerization to the poly- meric state of molecular weight 10 X lo6 (31). The present studies were designed after our earlier investigations of the effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the acetyl-CoA carboxylase (31, 46). In this report we present our results on the changes in these properties of the enzyme taking place in uiuo after short-term administration of insulin, glucagon, or epinephrine to rats. Our results show that insulin causes activation of the hepatic acetyl-CoA carboxylase with a lower citrate require- ment and dephosphorylation and polymerization of the pro- tein. Glucagon or epinephrine administration, on the other hand, lowers carboxylase activity, increases the phosphate content, and depolymerizes the protein.

EXPERIMENTAL PROCEDURES

Materials-HumulinTM R and glucagon were gifts from Lilly. NovolinTM R was purchased from Squibb Novo, Inc.. and eDineDhrine (AdrenalinTM chldride solution) fro& Parke-D&is. ‘?he so;rces of all other materials were described previously (46). Sprague-Dawley rats (retired female breeders) weighing 200-300 g each were purchased from Harlan Sprague-Dawley, Inc.

Animals, Hormone Administration, and Blood Level Analvsis-Rats were divided into two groups with 14 animals per group. One group was injected with the indicated hormone and the other with an equal volume of saline as a control. The first two groups were fed a normal laboratory diet and injected intraperitoneally with either insulin or saline. To study the proposed activation of acetyl-CoA carboxylase

by insulin, the logical choice would have been fasted animals where carboxylase is known to be relatively inactive. However, due to low levels of blood glucose in the fasted animals, insulin administration was not possible because of accompanying “insulin shock.” For this reason, animals on a normal laboratory diet were chosen.

To study glucagon-induced or epinephrine-induced inactivation of carboxylase, if any, fasted and refed rats were chosen mainly because the carboxylase in such rats is known to be highly active (31). Thus, another two groups of rats were fasted for 2 days and refed a high- carbohydrate, low-fat diet for 2 days. After a 2-h deprivation of food, these animals were injected intraperitoneally with either glucagon or saline. This short period of deprivation of food was introduced to guard against high levels of blood glucose that would cause the release of endogenous insulin which in turn would have antagonized the glucagon action. Similar groups of rats were maintained on the same regimen and used for the epinephrine experiment.

About 10 min after hormone administration blood was drawn from the hearts of two rats from each of the groups. The blood glucose level was determined using a Sigma diagnostic kit (Trinder). The insulin and glucagon levels in the blood were determined by radio- immunoassay (47). The remaining 12 rats from each group were anesthetized with pentobarbital (60 mg/kg; NembutalTM sodium so- lution, Abbott) and the livers were excised and freeze-clamped as described previously (46). The frozen livers were pulverized in liquid nitrogen and stored at -70 “C! until used.

Preparation of Carboxylase and Superose 6 Gel Permeation-Ace- tyl-CoA carboxylase was purified from frozen liver powder as de- scribed by Thampy and Wakil (46). The polymeric and octameric forms of the enzyme were separated by using a Superose 6 HR column (1.0 X 30 cm) from Pharmacia LKB Biotechnology Inc. operated with an LKB GTi high-pressure liquid chromatography system. The col- umn was equilibrated at room temperature with 20 mM Tris-HCl, pH 7.2, containing 1 mM EDTA and 100 mM NaCl. About 300-400 pg of acetyl-CoA carboxylase was applied onto the column in a volume of 200 11 and the proteins were eluted with the same buffer at a rate of 0.5 ml/min (46).

Dephosphorylation and Polymerization of Acetyl-CoA Carboxyluse by [Acety-CoA Carboxylusel-phosphntase P-Acetyl-CoA carboxylase was incubated with purified phosphatase for 20 min at 37 “C! at a ratio of 2:l (w/w) in a reaction mixture similar to that described earlier (31, 46). The reaction was stopped by cooling on ice and an aliquot of 200 ~1 was applied immediately onto a Superose 6 column and the enzyme was eiuied as described above. Another aliquot was assayed simultaneously for acetyl-CoA carboxvlase activity. The re- maining mixture cant-tining lob pg of acetyl-CoA carboxylase was dialyzed against 2 liters of 100 mM Tris-HCl, pH 7.0, containing 10% glycerol and 1 mM EDTA to remove the inorganic phosphate gener- ated by the phosphatase action. The dialyzed enzyme was then treated with 1.0 N NaOH and the liberated phosphate was determined as described below.

Protein Determination-The bicinchoninic acid method (modified Lowry) (56) was used according to the manufacturer’s (Pierce Chem- ical Co.) recommendations to determine the protein concentration of the purified acetyl-CoA carboxylase. Bovine serum albumin was used as a standard.

Acetyl-CoA Carboxyluse Assay-The carboxylase was assayed by measuring the incorporation of [“Clbicarbonate into malonyl-CoA (13). Each assay tube contained 0.05-0.2 pg of affinity-purified en- zyme in a final volume of 0.15 ml. One unit of activity is defined as 1 pmol of malonyl-CoA formed per min at 37 “C. The specific activity is defined as units/mg of protein.

Phosphate Determination-Affinity-purified carboxylase (20-40 pg) was hydrolyzed in 1.0 N NaOH (final volume 0.1 ml) at 80 “C for 4 h. The hydrolysate was cooled to room temperature, acidified with 2 eq of sulfuric acid, and the phosphate was determined colorimetri- tally by the addition of 1.0 ml of reagent as described earlier (48). The intensity of color was measured at 660 nm using inorganic phosphate as a standard.

[Acetyl-CoA Carboxykwel-phosphatase 2 Assay-The phosphatase was assayed by measuring the rate of activation of affinity-purified acetyl-CoA carboxylase as described earlier (13, 46), except that the Mn2+ concentration was increased to 3.0 mM.

Purification of [Acetyl-CoA Carboxylasel-phosphatase 2-The su- pernatant fluid (750-800 ml) obtained from the second (5.5%) poly- ethylene glycol precipitation step during the purification of the a,$- CoA carboxylase (46) from insulin-treated rats was diluted I-fold with distilled water and applied dropwise onto a DEAE-Bio-Gel column (300 ml in a column (9.0-cm diameter X 5.0-cm height))

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6332 Hormonal Control of Acetyl-CoA Carboxylase

equilibrated with 20 mM Tris-HCl, pH 7.1 (buffer A). All steps were carried out at 4 “C. The column was washed with buffer A until no 280 nm-absorbing materials were released. The proteins were then eluted from the DEAE-Bio-Gel with buffer A containing 0.5 M NaCl. The eluate was mixed with 50 ml of avidin-Sepharose and left to stand for 1.5 h with gentle agitation to remove any acetyl-CoA carhoxylase remaining in the supernatant fluid. The avidin-Sepharose was removed by filtration and washed with 100 ml of buffer A containing 0.5 M NaCl. The proteins from the wash and the filtrate were precipitated by ammonium sulfate at 50% saturation. The precipitated proteins were removed by centrifugation and dissolved in buffer A, dialyzed against the same buffer for 2-3 h, and applied onto a DEAE-Bio-Gel column (3 x 10 cm) equilibrated with buffer A. The flow-through solution was collected and reapplied onto the column to ensure total absorption of the phosphatase. The DEAE- Bio-Gel was then washed with buffer A until no 280 nm-absorbing materials were released. The column was washed with 100 ml of buffer A containing 100 mM NaCl, then followed with a NaCl gradient made up from 300 ml each of 100 mM and 250 mM NaCl in buffer A. Fractions of 4.0 ml were collected and assayed for acetyl-CoA carbox- ylase phosphatase activity. The fractions exhibiting phosphatase activity were pooled and the proteins were precipitated with ammo- nium sulfate at 50% saturation. The precipitate was removed by centrifugation, dissolved in buffer A (50-100 ml), diluted 20 times with buffer A, and applied onto a Q-Sepharose Fast Flow column (3 x 10 cm) pre-equilibrated with buffer A. The column was washed with buffer A until no more proteins were detected in the wash, followed by another wash with 100 ml of buffer A containing 250 mM NaCl. The phosphatase was then eluted with a linear gradient of 100 ml each of buffer A containing 250 and 500 mM NaCl. Fractions (3 ml) were collected and assayed for the phosphatase. Those fractions containing the highest activity were pooled and the proteins were precipitated with ammonium sulfate at 50% saturation. The precipi- tated proteins were removed by centrifugation and dissolved in buffer A containing 1 mM EDTA and 100 mM NaCl (buffer B) and applied onto a Superose 12 HR lo/30 gel-filtration column, pre-equilibrated with buffer B and connected to an LKB fast-protein liquid chroma- tography system. The proteins were eluted with buffer B and the fractions with the highest activity were pooled and the proteins precipitated with ammonium sulfate at 50% saturation. The precipi- tate was removed by centrifugation, dissolved in buffer B, and stored at -70 ‘C.

RESULTS

Intraperitoneal administration of insulin to rats resulted in rapid hypoglycemia and a marked increase in the serum insulin/glucagon molar ratio (Table I). Injection of glucagon, on the other hand, had an opposite effect, resulting in hyper- glycemia and a sharp decrease in the insulin/glucagon ratio. Administration of epinephrine resulted in an increase in the level of blood glucose as shown in Table I. Thus, having

demonstrated that the injected hormones were active in mod- ifying blood glucose levels within the first 10 min after their introduction, we proceeded to explore the effect of these hormones on liver acetyl-CoA carboxylase.

Purity of Acetyl-CoA Carboxylase-The enzyme purified by avidin-Sepharose chromatography from rapidly freeze- clamped livers of rats appeared as a major protein band (290%) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with no evidence of significant proteolysis (Fig. 1). The various preparations of acetyl-CoA carboxylase appeared indistinguishable based on their electro- phoretic mobilities on such gels. The minor protein present is usually pyruvate carboxylase (&fr 125,000), which in our experience does not affect the assays. Using the chicken liver fatty acid synthase (M, 262,000), myosin (Mr 215,000), and pyruvate carboxylase (Mr 130,000) as standards, the subunit molecular weight of the carboxylase protein band was calcu- lated to be 260,000, which is close to the calculated molecular weight of 265,220 (49) based on cDNA sequencing and the predicted amino acid sequence of 2,345 amino acid residues.

Effect of Insulin Administration on Actiuity, Phosphate Con- tent, and Polymerization State of Carboxylase-Acetyl-CoA carboxylase purified from freeze-clamped livers of rats fed a normal laboratory diet possessed an activity of 0.8 units/mg when assayed in the absence of citrate. Addition of citrate resulted in stimulation of this activity, with half-maximal activation (K& observed at a concentration of 1.0 mM citrate (Fig. 2). Although this citrate concentration is slightly higher than physiological levels, it is significantly lower than that reported for acetyl-CoA carboxylase prepared from unfrozen tissues (13, 16, 17).

Injection of insulin into animals fed a normal laboratory diet resulted in increased activity of the purified acetyl-CoA carboxylase (Fig. 2). The increases in enzyme activity were noted both in the absence and presence of citrate. However, the addition of citrate further stimulated the activity of the carboxylase, with half-maximal activation observed at about 0.6 mM citrate, which is near its physiological concentrations. This activation was insulin dose-dependent and a maximum 2.5-fold increase in specific activity of the carboxylase was observed after injection of 5 units of insulin per animal (Fig. 3).

Analyses of the carboxylase for alkali-labile phosphate con- tent showed that the enzyme prepared from animals that did not receive insulin had a relatively high phosphate content

TABLE I Effect of hormones on blood glucose level and acetyl-CoA carboxylase activity

The blood glucose level and the immunoreactive insulin and glucagon were determined as described under “Experimental Procedures.” The mean + S. E. of 10 blood samples representing five preparations are shown. The percentage of the polymeric form of the carboxylase was estimated from the areas under the peaks plotted from the chromatogram of the Superose 6 column.

Animal treatment Carboxylase Blood glu- Insulin/glucagon

Diet Hormone injection case level molar ratio (per rat) Activity PolymeP

mg/100 ml unitsfmg %

Laboratory Insulin None 148 -c 5 9f2 0.8 f 0.1 8 5 units (0.19 mg) 50 f 3 804 f 100 2.1 f 0.1 56

Fasted/refed Glucagon None 118 + 0.2 12 zk 3 3.4 k 0.1 68 1 unit (1.0 mg) 200 f 6 0.04 f 0.01 1.4 + 0.04 24

Epinephrine None 120 3.4 1 w 204 1.9

a Molecular weight 10 x 106.

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FIG. 1. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis of acetyl-CoA carboxylase prepared from animals after treatment with insulin or glucagon. Lanes 1 and 2 contain enzymes (2 rg) purified from livers of rats fed a laboratory diet and injected with saline or insulin, respec- tively; lanes 3 and 4 contain enzymes (3 rg) purified from livers of fasted-refed rats injected with saline or glucagon, re- spectively; and lanes labeled with S con- tain proteins as molecular weight mark- ers (myosin, 215,000; macroglobulin, 170,000; P-galactosidase, 116,000; trans- ferrin, 76,000; glutamic dehydrogenase, 53,000).

Hormonal Control of Acetyl-CoA Carboxylase

; 1 2

. i

4#k

0 5 10

Citrate [mM]

FIG. 2. Citrate dependence of preparations of acetyl-CoA carboxylase isolated from livers of rats injected with insulin or saline. Acetyl-CoA carboxylase was assayed at the indicated citrate concentrations as described under “Experimental Procedures.” The enzyme was prepared from rats maintained on a laboratory diet and injected with saline (A) or 5 units (-0.19 mg) of insulin (A) prior to extraction of the livers. The same carboxylase preparations from rats injected with saline (0) or insulin (e), respectively, were treated with the [acetyl-CoA carboxylasel-phosphatase 2 as described under “Experimental Procedures” and assayed at the indicated citrate con- centrations. Points, mean; vertical bars, + S. E.

comparable to that of the carboxylase isolated from unfrozen livers (46). Insulin administration led to a net decrease in the phosphate content of the carboxylase from 8.5 to 7.0 ml of Pi/m01 of subunit (Table II).

The enzyme preparation from animals that did not receive insulin eluted from the Superose 6 column in two fractions.

3 4 s

6333

I I I I I

0 2 4

Insulin [units]

FIG. 3. Activation of acetyl-CoA carboxylase in response to insulin injections. Carboxylase was isolated from livers of rats maintained on a normal laboratory diet and injected intraperitoneally with the indicated doses of insulin. Preparations and assay of carbox- ylase were performed as described under “Experimental Procedures.”

A minor fraction, obtained in 8 ml of eluate, represents proteins with an estimated molecular weight of 10 X lo6 (polymeric state) and a major fraction, collected in about lo- 12 ml of eluate, represents protein with an estimated molec- ular weight of 2 X lo6 (octameric state; Fig. 4B). Insulin administration, however, led to a pronounced increase in the polymeric form of the enzyme as shown by the shift from the octameric to the polymeric form in the elution profile (Fig. 4C).

The insulin-induced activation of acetyl-CoA carboxylase, its polymerization, and its higher citrate sensitivity may be attributed to its lower phosphate content since dephosphory- lation of the enzyme isolated from rats that did not receive insulin by [acetyl-CoA carboxylasel-phosphatase 2 resulted in similar transformations in activity of the carboxylase, its response to citrate, and its polymeric states (Figs. 2 and 4). The enzyme isolated from insulin-treated animals can undergo further polymerization when treated with [acetyl- CoA carboxylasel-phosphatase 2 as evidenced by the complete

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6334 Hormonal Control of Acetyl-CoA Carboxylase

TABLE II

Effect of hormones (insulin, glucagon, or epinephrine) on activity and phosphate content of acetyl-CoA carboxylnse All the values given below represent the mean f S. E. from four preparations, except for those obtained after

treatment of the carboxylase with phosphatase. For the enzyme prepared from insulin-, glucagon-, or epinephrine- injected animals, the values represent the means of two, four, and two determinations, respectively.

Source of carboxylase preparations Specific activity

Animal maintenance Phosphatase & citrate Phosphate content

Diet Hormone in- treatment* Plus citrate

iection” No citrate

(10 nlM)

units/mg mhf mol P&no1 subunit

Laboratory Saline No 0.8 + 0.13 3.8 f 0.3 1.0 + 0.04 8.5 f 0.3 Insulin No 2.1 * 0.07 4.8 + 0.1 0.6 k 0.02 7.0 f 0.3 Insulin Yes 9.0 + 0.27 12.5 + 0.25 0.17 f 0.05 3.65 + 0.3

Fasted/refed’ Saline No 3.4 + 0.11 7.7 + 0.5 0.2 + 0.03 4.9 + 0.5 Glucagon No 1.4 f 0.04 5.0 + 0.2 1.0 + 0.06 6.4 + 0.5 Glucagon Yes 8.1 + 0.01 9.7 + 0.1 0.17 -c 0.05 5.2 f 0.4 Epinephrine No 1.9 5.4 1.0 + 0.1 6.7 k 0.2

’ Five units of insulin, 1 unit of glucagon, or 1 mg of epinephrine were injected intraperitoneally as indicated and the animals were killed 10 min later.

* Where indicated, carboxylase was treated with phosphatase as described under “Experimental Procedures.” ’ Food was withdrawn from these rats 2 h before injection with hormones.

conversion of the protein to the polymeric form as noted in the elution profile from the Superose 6 column (Fig. 40). These changes were also accompanied by significant increases in the citrate-independent and citrate-dependent activities of the carboxylase (Fig. 2).

Glucagon-induced Phosphorylation and Inhibition of Car- box&se-In agreement with previous reports (9), acetyl-CoA carboxylase purified from freeze-clamped livers of fasted and refed rats exhibited high, citrate-independent activity (3.5 units/mg), with a low & value for citrate activation and low phosphate content. Treatment of similarly fasted and refed animals with glucagon prior to extraction of the livers yielded enzyme preparations with relatively lower activity (60% de- crease in specific activity), a &fold increase in Koa values for citrate activation, and a higher phosphate content (Table II). Moreover, the specific activity of the carboxylase was lower at all citrate concentrations tested as shown in Fig. 5. Treat- ment of this carboxylase with [acetyl-CoA carboxylasel-phos- phatase 2 increased significantly the citrate-independent ac- tivity as shown in Fig. 5.

Chromatography of these preparations on a Superose 6 column gave elution patterns suggesting that the enzyme from fasted/refed animals is comprised of two polymer species, a major one (Mr - 10 X 106) and a minor one (Mr = 2 X 106; Fig. 6A). Glucagon treatment of similarly fasted and refed rats yielded an enzyme preparation with an elution pattern from the Superose 6 column significantly different from the enzyme isolated from saline-injected animals. Glucagon in- creases the relative content of the octameric form (Fig. 6B). Dephosphorylation of this carboxylase preparation by [acetyl- CoA carboxylasel-phosphatase 2 resulted in significant acti- vation of the carboxylase with a concomitant decrease in the & value for citrate (Fig. 5) and polymerization of the protein (Fig. 6C), suggesting that these changes are due to phos- phorylation of the carboxylase subunit.

Effect of Epinephrine on Acetyl-CoA Carboxylose-Like glu- cagon, injection of epinephrine into fasted/refed rats resulted in a decrease in carboxylase activity and a 5-fold increase in & values for citrate activation (Table II). Chromatography of the carboxylase preparations on a Superose 6 column gave elution profiles similar to those obtained with carboxylase preparations isolated from the group of rats injected with glucagon (Fig. 7). Treatment of the carboxylase with citrate resulted in activation and polymerization of the enzyme (Fig.

7C). The carboxylase was similarly activated and polymerized when treated with [acetyl-CoA carboxylasel-phosphatase 2 (data not shown).

DISCUSSION

The acetyl-CoA carboxylase of animal tissues is a complex multifunctional enzyme. The subunit protein is large (Mr - 265,000), containing a biotin-binding site, the catalytic do- mains for the carboxylation of biotin and transfer of the carboxyl group to acetyl-CoA, and the regulatory sites for the binding of allosteric effecters, citrate and palmitoyl-CoA, and numerous phosphorylation sites. The enzyme also tends to aggregate, especially after activation of the carboxylase, form- ing polymers of up to 10 X lo6 in molecular weight (10, 13, 14, 19, 31). Moreover, the protein subunit is susceptible to proteolysis which also results in modification of activity (50). In recent studies of the acetyl-CoA carboxylase, we related citrate sensitivity, phosphorylation, and polymerization to each other and presented evidence that the enzyme undergoes modifications during its purification from animal sources (31). For instance, in the crude stages of preparation the enzyme may undergo proteolysis, rapid phosphorylation (51) (presum- ably using cellular ATP and endogenous kinases), and de- phosphorylation catalyzed by endogenous phosphatases (13, 52). While proteolysis can have a minor effect on catalytic activity and may be contained by the use of protease inhibi- tors, changes in the phosphorylation state affected activity, citrate dependence, and polymerization of the protein (31). Consequently, we developed a method of rapid purification of the enzyme by avidin-affinity chromatography after including a variety of protease inhibitors designed to minimize hydrol- ysis of the protein subunit in the medium (46). These methods led for the first time to the isolation of the active, citrate- independent, and polymeric form of the carboxylase from animal tissues (46). Moreover, the earlier isolation of a specific phosphatase, [acetyl-CoA carboxylasel-phosphatase 2, that activates the carboxylase and renders it citrate-independent made it possible to relate the phenomenon of citrate activation to phosphorylation and polymerization of the enzyme (13, 31). Based on these observations we then studied the inter- relationships of changes in specific catalytic activity, citrate dependence, phosphate content, and polymerization state of the carboxylase as a function of fasting and refeeding (31). In the present investigation, we extended these previous studies

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Hormonal Control of Acetyl-CoA Carboxylase 6335

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FIG. 4. Elution profile from Superose 6 gel permeation col- umn of acetyl-CoA carboxylase prepared from animals in- jected with insulin. Carboxylase preparations used were purified by affinity chromatography from freeze-clamped livers of rats fed a laboratory diet and injected with either saline or insulin 10 min before killing. 1, point of application of the samples onto the column. A, elution profile of a mixture of known proteins used as a reference standard (peak 1, pyruvate dehydrogenase, M, 10 x 106; peak IZ, yeast fatty acid synthase, M, 2.4 X 106; peak III, rat liver fatty acid synthase, M, 525,000; peak IV, ferritin, M, 440,000; peak V, bovine serum albumin, M, 63,000); inset, linear plot of molecular weight uersu.s elution volume; B, elution profile of acetyl-CoA carboxylase (400 rg) prepared from rats injected with saline; C, elution profile of acetyl- CoA carboxylase (350 pg) purified from livers of rats injected with 5 units of insulin; D, elution profile of acetyl-CoA carboxylase (90 rg) employed in the experiment represented in C, but after treatment with acetyl-CoA phosphatase as described under “Experimental Pro- cedures.”

I I 0 1 5 10

Citrate [mM]

FIG. 5. Citrate dependence of preparations of acetyl-CoA carboxylase isolated from livers of fasted/refed rats injected with saline or glucagon. 0 and 0, citrate activation of carboxylase preparations isolated from animals injected with saline or glucagon, respectively. A, shows citrate activation of carboxylase prepared from livers of animals injected with glucagon as that used in experiments depicted in curve 0, but with added treatment of the purified enzyme with [acetyl-CoA carboxylasel-phosphatase 2 prior to assaying as described under “Experimental Procedures.” Points, mean; vertical bars, f S. E.

to include the acute effects of insulin, glucagon, and epineph- rine on the properties of the carboxylase. This report repre- sents the first direct demonstration of the effects of these important hormones on the various properties of the highly purified acetyl-CoA carboxylase when they are administered to the whole animal.

The hormones insulin or glucagon were injected intraperi- toneally into rats and their effects on acetyl-CoA carboxylase and blood glucose levels were evaluated and related to both hormone dosage and the time lapsed before the animals were killed. The injection of 5 units of insulin causes a 70% decrease in the level of blood glucose, while the injection of 1 unit of glucagon results in a near doubling of glucose concentration (Table I). These levels of hormones were optimal for eliciting significant changes in blood glucose levels and the insulin/ glucagon ratios, and at the same time are tolerated by the animals. Also, changes in carboxylase activity are near max- imal at these levels (Fig. 3). Concomitant with changes in carboxylase activity, there is alteration in the polymeric form of the enzyme. Insulin injection resulted in polymerization of the carboxylase, while glucagon administration resulted in depolymerization of the protein (Table I; Figs. 4 and 6). It is important to note here that maintenance of the animals on the indicated diet (laboratory chow for animals used in the insulin experiments and starvation/refeeding with a high- carbohydrate diet for animals used in the glucagon experi- ments) is important for the success of these experiments. Acetyl-CoA carboxylase is an inducible enzyme whose amount and activity is regulated by diet. Hence, acetyl-CoA carbox- ylase is reasonably well expressed in rats maintained on a laboratory diet but has a relatively low activity, making this a suitable condition to test stimulation by insulin. Similarly, rats maintained on a high-carbohydrate diet after a period of starvation have a highly expressed and active carboxylase; glucagon administration causes a quantitatively significant

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6336 Hormonal Control of Acetyl-CoA Carboxyhe

0.0

0.02

0.0

E r

8 fi 0.02

a

i 2

0.0

0.0 ‘1 L I 0 4

L 3 12

0.025

0 0 4 0 12 1t

Elution Volume [ml]

Elution Volume [ml]

FIG. 6. Elution profile from Superose 6 gel permeation col- umn of acetyl-CoA carboxylase prepared from animals in- jected with glucagon. The carboxylase preparations used were purified from freeze-clamped livers of fasted-refed rats after injection with saline or glucagon as described under “Experimental Proce- dures.” J, points of application of the enzyme onto the column. A and B, elution profiles of acetyl-CoA carboxylase (400 rg) prepared from livers of rats injected with saline or glucagon, respectively; C, elution profile of acetyl-CoA carboxylase (90 pg) used in the experiment depicted in B, but treated with (acetyl-CoA carboxylasel-phosphatase 2 prior to its chromatography on a Superose 6 column,

decrease in activity (Table I). Administration of insulin acti- vated the carboxylase, concomitantly dephosphorylated it, increased its citrate sensitivity, and polymerized the protein. Thus, the short-term effects of insulin on acetyl-CoA carbox- ylase mimic those of refeeding (31).

While the effects of insulin on carboxylase were coordinated in the direction of increased enzyme activity, that of glucagon or epinephrine were in the opposite direction. Thus, the net glucagon-induced phosphorylation, depolymerization, and in- creased citrate dependence of acetyl-CoA carboxylase lowered its activity. These effects of glucagon on acetyl-CoA carbox- ylase are very similar to those observed after brief fasting (31). Therefore, the insulin- or glucagon-induced changes described here further support the hypothesis that acetyl-CoA carboxylase exists in two forms, an active, citrate-independent polymeric form and an inactive octamer, that are intercon- vertible by phosphorylation/dephosphorylation. These re- sults, together with earlier reports, clearly establish the con- cept that dephosphorylation of acetyl-CoA carboxylase leads to activation while phosphorylation leads to inactivation (13, 19, 31, 37, 51). This conclusion is also compatible with the general dogma that enzymes in the anabolic pathways are inhibited by phosphorylation and stimulated by dephospho- rylation.

FIG. 7. Elution profile from Superose 6 gel permeation col- umn of acetyl-CoA carboxylase prepared from animals in- jected with epinephrine. The carboxylase preparations used were purified from freeze-clamped livers of fasted/refed rats after subcu- taneous injection with saline or epinephrine as described under “Ex- perimental Procedures.” J, points of application of the enzyme onto the column. A and B, elution profiles of carboxylase (400 pg) prepared from the livers of rats injected with saline or epinephrine, respectively; C, elution profile of the carboxylase (400 pg) used in the experiment depicted in B, except that the enzyme was treated with 10 mM citrate prior to chromatography.

Earlier reports on the insulin-induced phosphorylation and activation of acetyl-CoA carboxylase (33, 34, 42-45) were in apparent contradiction with the aforementioned conclusion. These studies reported increased incorporation of 32P into the carboxylase protein. This may have been an artifact of isola- tion of the enzyme because acetyl-CoA carboxylase is rapidly phosphorylated immediately after excision of liver from the rat; a quick freeze-clamping of the tissue was necessary to preserve the enzyme in its active form (46). Thus, precaution- ary measures must be taken to avoid phosphorylation of the enzyme, which in addition to increasing phosphate content, may lead to inhibition of activity, increased citrate depend- ence, and depolymerization of the protein subunits, depending upon the sites phosphorylated. These changes may explain, at least in part, why past studies detected increased phos- phorylation of carboxylase without activation (32, 41), or phosphorylation and activation (33,34,42-44), or phosphoryl- ation, activation, and polymerization (45) in the presence of insulin.

Our results are in agreement with a recent report on the insulin-dependent activation and dephosphorylation of ace- tyl-CoA carboxylase (40) and a number of reports on the glucagon-induced inhibition and phosphorylation of the en- zyme (35, 37, 38). The major difference between the study presented here and those of other investigators is the consis- tent demonstration of activation, dephosphorylation, and po- lymerization as induced by insulin, and the inactivation,

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Hormonal Control of Acetyl-CoA Carboxylase 6337

phosphorylation, and depolymerization of carboxylase in af- finity-purified preparations as induced by glucagon or epi- nephrine when administered to whole animals. Our results imply that, for enzymes involved in the regulation of carbox- ylase, the ratio of phosphatase activity to kinase activity is modified by these hormones. These modifications may be achieved by selective activation of one or more of the phos- phatases, or inhibition of one or more kinases, or both. In the case of insulin action, we have detected a significant increase in the specific activity of [acetyl-CoA carboxylasel-phospha- tase 2 in purified fractions from livers of insulin-treated rats (data not shown); the involved mechanism is not clear. [Ace- tyl-CoA carboxylasel-phosphatase 2 may be regulated by phosphorylation, but supporting data are lacking.

Finally, the recent cloning and sequence analysis of the cDNA coding for the rat acetyl-CoA carboxylase made it possible to predict the amino acid sequence of the protein subunit (49). Assignment of the phosphorylation sites has also been made based on the amino acid sequence of the various phosphopeptides isolated from the carboxylase after treatment with different protein kinases under various in uiuo and in vitro conditions (30,42,43). The rat carboxylase cDNA codes for an open frame of 2345 amino acids with seven phosphorylation sites, six of which are located near the NH2 terminus at serine positions 23, 25, 29, 76, 77, and 95. The seventh site is located at serine residue 1200 (54). Although these sites may be phosphorylated by some known protein kinases in vitro, the identity and hormonal regulation of the protein kinases that carry out the phosphorylation in vivo are not known. Among the protein kinases that are thought to be involved in the phosphorylation and, hence, the regulation of the carboxylase are CAMP-dependent protein kinase (19), Ca2+-dependent protein kinase (19), CAMP-independent pro- tein kinase (25), AMP-dependent protein kinase (30), protein kinase C (42, 53, 54), calmodulin-dependent protein kinase (42), and casein kinase 2 (34). The inactivation of the carbox- ylase by glucagon and epinephrine is probably brought about by phosphorylation of the enzyme by CAMP-dependent pro- tein kinase or Ca*+-dependent protein kinase, respectively. Whether these two hormones exert their effects through the CAMP-independent protein kinase or AMP-dependent kinase remains to be determined. However, investigations of the effect of insulin in the activation of the carboxylase have been difficult, especially since the mechanism of action of this hormone is not yet known and its metabolic and cellular effects are proving to be more and more complex. In one way or another, several kinases (protein kinase C, casein kinase 2, and calmodulin-dependent protein kinase) have been impli- cated in the activation of carboxylase by insulin (36, 42, 53, 54). On the other hand, incubation of Swiss mouse 3T3-Dl cells with insulin rapidly and transiently activated a protein phosphatase (55), which led the authors to suggest that “some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.” Our results are consistent with this conclusion. Insulin may activate the carboxylase by dephosphorylating the enzyme. Whether this represents activation of a phospha- tase by insulin via reversible phosphorylation remains to be established. The Mn*+-requiring [acetyl-CoA carboxylase] - phosphatase 2 has a high affinity for the carboxylase and may be one such phosphatase. This phosphatase mimics the action of insulin, promoting activation and polymerization of the carboxylase and rendering it more sensitive to citrate.

Acknowledgments-We thank Dr. Aubrey E. Boyd, III and mem- bers of the Diabetes Core Facility at Baylor College of Medicine, for measurement and analysis of insulin and glucagon, and Pamela

Powell for editorial assistance during the preparation of this manu- script. One of us (I. M. H.) would like to thank Professor Salah Eid, Department of Biochemistry, Ain Shams University, Cairo, Egypt, for his support, encouragement, and advice, and Ain Shams Univer- sity and the Egyptian government for scholarship support.

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REFERENCES

Wakil, S. J. (1958) J. Am. Chem. Sot. SO,6465 Wakil, S. J., Titchener, E. B., and Gibson, D. M. (1958) Biochim.

Biophys. Acta 29, 225-226 Wakil, S. J., Stoops, J. K., and Joshi, V. C. (1983) Annu. Reu.

Biochem. 52,537-579 McGarry, J. D., Leatherman, G. F., and Foster, D. W. (1978) J.

Biol. Chem. 253.4128-4136 Bai, D. H., Pape, M. E., Lopez-Casillas, F., Luo, X. C., Dixon, J.

E., and Kim, K.-H. (1986) J. Biol. Chem. 261,12395-12399 Majerus, P. W., and Kilburn, E. (1969) J. Biol. Chem. 244,6254-

6262 Nakanishi, S., and Numa, S. (1970) Eur. J. Biochem. 16, 161-

173 Krakower, G. R., and Kim, K.-H. (1981) J. Biol. Chem. 256,

2408-2413 Hardie, D. G., and Cohen, P. (1979) FEBS Lett. 103,333-338 Lane, M. D., Moss, J., and Polakiss, E. (1973) Curr. Top. Cell.

Regul. 8, 139-195 Pekala, P. H., Meredith, M. J., Tarlow, D. M., and Lane, M. D.

(1978) J. Biol. Chem. 253,5267-5269 Beaty, N. B., and Lane, M. D. (1983) J. Biol. Chem. 258,13043-

13050 Thampy, K. G., and Wakil, S. J. (1985) J. Biol. Chem. 260,

63186323 Lane, M. D., Watkins, P. A., and Meredith, M. J. (1979) CRC

Crit. Reu. Biochem. 7, 121-141 Lowenstein, J. M. (1968) in The Metabolic Roles of Citrate (Good-

win, T. W., ed) pp. 61-68, Academic Press, New York Wakil, S. J., and Barnes, E. M., Jr. (1971) in Comprehensive

Biochemistry (Florkin, M., and St&z, E. M., eds), Vol. 185, pp. 57-104, Elsevier Science Publishing Co., New York

Kim, K.-H. (1979) Mol. Cell. Biochem. 28, 27-43 Siess, E. A., Brocks, D. G., and Wieland, 0. H. (1978) Hoppe-

Seyler’s Z. Physiol. Chem. 359, 785-798 Kim, K.-H. (1983) Curr. Top. Cell. Regul. 22, 143-176 Witters, L. A., and Vogt, B. (1981) J. Lipid Res. 22, 364-369 Ahmad, F., and Ahmad, P. M. (1981) Methods Enzymol. 71, 16-

26 Hardie, D. G., and Cohen, P. (1978) FEBS I&t. 91, l-7 Tipper, J. P., and Witters, L. A. (1982) Biochim. Biophys. Acta

715.162-169 Wada, K., and Tanabe, T. (1983) Eur. J. Biochem. 135,17-23 Lent, B., and Kim, K.-H. (1982) J. Biol. Chem. 257,1897-1901 Lent, B. A., and Kim, K.-H. (1983) Arch. Biochem. Biophys. 225,

972-978 Hardie, D. G. (1980) in Molecular Aspects of Regulation (Cohen,

P., ed) Vol. 1, pp. 33-62, Elsevier, Amsterdam Hardie, D. G., and Guy, P. S. (1980) Eur. J. Biochem. 110, 167-

177 Carling, D., Zammit, V. A., and Hardie, D. G. (1988) FEBS Z&t.

223; 217-222 Munday, M. R., Campbell, D. G., Carling, D., and Hardie, D. G.

(1988) Eur. J. &o&m. 175. 331-338 Thampy, K. G., and Wakil, S. J. (1988) J. Biol. Chem. 263,

6454-6458 Witters, L. A. (1981) Biochem. Biophys. Res. Commun. 100,872-

878 Brownsey, R. W., and Denton, R. M. (1982) Biochem. J. 202,

77-86 Witters, L. A., Tipper, J. P., and Bacon, G. W. (1983) J. Biol.

Chem. 258,5643-5648 Holland, R., Witters, L. A., and Hardie, D. G. (1984) Eur. J.

Biochem. 140,325-333 Denton, R. M. (1986) Adu. Cyclic Nucleotide Protein Phosphoryl-

ation Res. 20, 293-341 Sim, A. T. R., and Hardie, D. G. (1988) FEBS Z&t. 233, 294-

298 Swensen, T. L., and Porter, J. W. (1985) J. Biol. Chem. 260,

3791-3797

by guest on October 29, 2014

http://ww

w.jbc.org/

Dow

nloaded from

6338 Hormonal Control of Acetyl-CoA Carboxylase

39. Witters, L. A., Moriarity, D., and Martin, D. B. (1979) J. Biol. Chem. 254,6644-6649

40. Witters, L. A., Watts, T. D., Daniels, D. L., and Evans, J. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5473-5477

41. Holland, R., and Hardie, D. G. (1985) FEBS L&t. 181,308-312 42. Haystead, T. A. J., and Hardie, D. G. (1988) Eur. J. Biochem.

175,339-345 43. Haystead, T. A. J., and Hardie, D. G. (1986) Biochem. J. 240,

99-106

M. A., and Kim, K.-H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5784-5488

50. Numa, S., and Tanabe, T. (1984) in Fatty Acid Metabolism and Its Regulation (Numa, S., ed) pp. l-27, Elsevier, Science Pub- lishers, New York

51. Hardie, D. G., and Cohen, P. (1978) FEBS Z&t. 91, l-7 52. Thampy, K. G., and Wakil, S. J. (1986) Adu. Protein Phosphatases

3,257-269 53. Kim, K.-H., Lopez-Casillas, F., Bai, D. H., Luo, X., and Pape, M.

44. Zammit, V. A., and Corstophine, C. G. (1982) Biochem. J. 208, E. (1989) FASEB J. 3,2250-2256 783-788 54. Vaartzes, W. J., de Haas, C. G. M., Gellen, M. J. H., and Bijleveld,

45. Borthwick, A. C., Edgell, N. J., and Denton, R. M. (1987) C. (1987) Biochem. Biophys. Res. Commun. 142, 135-140 Biochem.J. 241,773-782 55. Chan, C. P., McNall, S. J., Krebs, E. G., and Fischer, E. H. (1988)

46. Thampy, K. G., and Wakil, S. J. (1988) J. Biol. Chem. 263, Proc. Natl. Acad. Sci. U. 5’. A. 85,6257-6261 6447-6453 56. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,

47. Morgan, C. R., and Lazarow, A. (1963) Diabetes 12, 115-126 Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. 48. Hess, H. H., and Derr, J. E. (1975) Anal. Biochem. 63,607-613 M., Olson, B. J., and Klenk, D. C. (1985) Anal. B&hem. 150, 49. Lopez-Casillas, F., Bai, D.-H., Luo, X., Kong, I.-S., Hermodson, 76-85

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http://ww

w.jbc.org/

Dow

nloaded from